Abstract TET2 is one of the most commonly mutated/deleted genes in adult hematological malignancies. TET2 mutations are also prevalent in healthy elderly individuals with clonal hematopoiesis. Thus, TET2 mutations are an ancestral mutational event that drives non-malignant clonal outgrowth and facilitates hematological malignancy transformation. Indeed, Tet2 loss in mice leads to increased HSC self-renewal and the development of various hematological malignancies. However, the underlying molecular mechanisms remain largely unknown. TET2 is a dioxygenase that catalyzes the stepwise conversion of 5mC to 5hmC, 5fC and 5caC, initial steps of active DNA demethylation. The oxidation and demethylation of 5mC in the genome are regulated in a sophisticated manner. It has been shown that 5hmC and 5fC are present as relatively stable cytosine modifications in genomic DNA of both dividing and nondividing cells. TET2 likely requires its catalytic activity to exert tumor suppressive function in HSC/HPCs. We recently showed that Tet2 loss leads to hypermutagenicity in HSC/HPCs, preferentially at genomic sites that gained 5hmC and TET2 normally binds to. TET2 loss would naturally remove part of, but also creates a new set of, stable 5hmC and 5fC marks in genomic DNA for an extended period in HSC/HPCs. However, the physiological significance of the TET2 loss-mediated stalling of 5hmC/5fC formation in HSC/HPC regulation and pathogenesis of hematological malignancies remains to be elucidated. We have created two novel Tet2 5hmC stalling (T1285E, Tet2E/+) and Tet2 catalytic-inactive (H1295Y/D1297A, Tet2YA/+) mutant knock-in mouse models, which provide us unique tools to elucidate the specific biological role of TET2 catalytic activity and TET2-dependent 5hmC?5fC conversion in HSC/HPC regulation and hematological malignancies. In Aim 1, we will elucidate the biological role of Tet2 loss-associated stalling of 5hmC and 5fC/5caC formation in HSC/HPC regulation and hematological malignancies using the catalytic-inactive and 5hmC stalling Tet2 mutant mouse models. In Aim 2, we will determine the effects of TET2 enzymatic activity and specific TET2-dependent DNA demethylation intermediates on gene expression regulation and genomic mutagenicity in HSC/HPCs. Using WT, Tet2-/-, Tet2YA/YA and Tet2E/E HSC/HPCs, we will: (1) perform RNA-seq to identify the differentially expressed genes (DEGs); (2) map genome-wide 5mC/5hmC/5fC/5caC marks; and (3) perform whole-exome sequencing to identify spontaneous mutations. Integrational analysis of these data sets will allow us to determine whether the DEGs and mutations caused by loss of TET2 catalytic activity or 5hmC?5fC conversion correlate with specific 5mC/5hmC/5fC/5caC alterations in HSC/HPCs upon Tet2 loss. These studies could unveil potential roles of specific TET2-dependent cytosine species in: (1) gene regulation and genomic mutagenicity in HSC/HPCs; (2) TET2 loss-mediated HSC/HPC dysregulation and hematological malignancies. These findings will greatly impact on the identification of novel therapeutic strategies for TET2-mutated hematological malignancies.